Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS–BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

of the intermolecular binding interactions and by disrupting the tertiary protein structure that form the binding domains 15,16 .
In addition, rapid screening tools to assess PFAS-protein binding are sorely needed.The most common methods used to probe this interaction are fluorescence spectroscopy, equilibrium dialysis, and fluorine nuclear magnetic resonance spectroscopy [17][18][19][20][21][22][23] .Although these techniques are widely used, they are also time consuming.While there are over 4,000 different compounds in the PFAS class, rapid and high throughput methods are essential for screening.
Recently, Differential Scanning Fluorimetry (DSF) has been proposed as a rapid, high throughput screening technique to quantify protein binding of various ligands, including drugs and small molecules 24,25 .DSF works by monitoring a change in fluorescence emission as a function of temperature.The most common method involves the use of a hydrophobic dye or "label" that fluoresces when bound to unfolded (denatured) protein 26 .Jackson et al. used DSF to investigate the binding of a wide range of legacy and emerging PFAS to human serum albumin (HSA) using the dye GloMelt™ as an indicator. 27For BSA, a label free option exists, as the tryptophan amino acid residue in the binding site is itself fluorescent and thus can be used as the fluorophore 28 .This technique reduces the potential for confounding effects from extrinsic fluorophores that may interfere with ligand binding or influence protein unfolding 29 .Based on molecular docking studies, PFOA and PFOS bind to BSA near tryptophan residue TRP-237, which was reported to be in close proximity to both PFAS bound at Sudlow site I (PFOA) 17,30 and site II (PFOS) 17 .As this binding event occurs, the proximity of the ligand to the fluorophore induces fluorescence quenching, which can be monitored by the change in fluorescence emission.This principle has previously been used by our group and others in fluorescence spectroscopy-based investigations of PFAS binding to BSA 21 .
In this work, PFAS binding to BSA was examined using DSF from 35 to 95 °C to inform future approaches to PFAS remediation from bovine milk, particularly those that would benefit from reduced protein binding affinity at elevated temperatures.The PFAS examined include common perfluorocarboxylic acids (PFCAs; PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoro-2-methyl-3-oxahexanoic acid (GenX)) and perfluorosulfonic acids (PFSAs; perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), and PFOS), as well as three fatty acids analogs (octanoic acid (OA), nonanoic acid (NA), and decanoic acid (DA)) that have the same carbon chain length as the PFCA (Table 1).Warfarin, a drug compound known to bind strongly to serum albumin protein at Sudlow site I 33 , was used as a control.The label-free approach was used to determine the effect of PFAS binding on the protein denaturing or melting temperature (T m ) and the PFAS-BSA association constant, K a , based on the Hill adsorption model.This model has proven more versatile than the classic Stern-Volmer quenching model and provides additional insight into protein-PFAS binding, such as binding cooperativity 21,34 .

Results and discussion
Results from DSF are demonstrated in Fig. 1. Figure 1a depicts the analysis approach where vertical transects were used to determine K a (approximated as K Hill , Fig. 1b) at a given temperature based on fluorescence quenching and the melting region associated with BSA unfolding was used to determine K a , which is inversely proportional to the dissociation constant K d , as well as the BSA melting temperature, T m (Fig. 1c).PFAS or FA binding did not shift the maximum fluorescence emission wavelength, indicating that fluorescence intensity was not influenced by ligand concentration for a given temperature.All the compounds studied resulted in fluorescence quenching, indicating that they all bind to BSA and lead to changes in tryptophan fluorescence.This is depicted in Fig. 1d-f for NA, PFNA, and PFOS; compounds with the same number of carbons, but with different headgroups (carboxylic acids NA, PFNA; sulfonic acid PFOS) and alkyl (NA) or fluoroalkyl (PFNA, PFOS) tails.

Quantitative analysis of binding constants at T m
We first determined the equilibrium-based binding constant evaluated near the melting temperature of pure BSA from the DSF curves (e.g., Fig. 1d-f).This method has been reported to yield a more accurate binding constant because it relies upon a simple model of coupled equilibrium states for both the protein and the ligand that accounts for protein folding and ligand binding 29 .The underlying assumption is that a ligand binds to the folded protein and not to the unfolded protein.Table 2 summarizes the association constants calculated at a temperature of 61 ºC.This temperature were selected because there were measurable differences between the fraction of unfolded protein for all of the PFAS and fatty acid complexes over the range of ligand:protein molar ratios, which is essential to applying Eqs. ( 1) and ( 2) to determine K d = K a −1 (see "Materials and methods" section).www.nature.com/scientificreports/Differences in the faction of unfolded protein, f U , for PFNA are shown in Figure S1 as a function of temperature for the different PFNA:BSA ratios.In this example, if a temperature below 61 °C was selected it would precede BSA melting at the highest PFNA:BSA ratio ( f U ≈ 0) and if a temperature above 63 °C was selected it would exceed BSA melting and the lowest PFNA:BSA ratio ( f U ≈ 1).Warfarin was used as a positive control and its K a agrees with values reported in the literature. 35It should be noted that K a for PFNA and PFHxS exceed that of warfarin, which was designed as a pharmaceutical drug to bind to BSA.We then examined the stabilizing effect of PFAS or FA on BSA structure based on increases in the protein melting temperature, ΔT m 36,37 .All PFAS compounds stabilized the folded structure of BSA, requiring greater thermal energy for protein melting with increasing PFAS concentration (Fig. 2).Oleic and nonanoic acids also stabilized the folded protein.There was a positive qualitative relationship between compounds that stabilized the folded protein and K a .PFNA, PFHxS, and NA had the greatest stabilizing effect and the highest K a values for their chemical group, while GenX and PFBS led to little stabilization with low K a values.The most effective stabilizing PFAS were PFOA, PFNA, and PFHxS, resulting in T m values ranging from roughly 69 to 71 °C whereas the native protein T m is 62 °C.Temperatures exceeding 69 °C to 71 °C would be required to desorb PFAS from BSA (e.g., during pasteurization).
Ligand binding is determined by the intermolecular interactions with the protein binding pocket and the size of the ligand.A compound may be very hydrophobic (high Log K ow ), which would lead to strong interactions within the hydrophobic binding pocket but may exhibit low binding if it is large and sterically hindered from fitting within the pocket.This is observed for PFDA, which has the highest Log K ow of the PFAS examined but showed the lowest K a with no measurable protein stabilization.K a values were plotted a function of Log K ow (predicted values, Table 1; EPA CompTox Chemicals Dashboard 31,32 ) and the number of fluorinated or hydrogenated carbons (Fig. 3).An inverted "V" shaped trend is observed for all compound classes (PFCA, PFSA, FA) with PFNA, PFHxS, and NA exhibiting the greatest binding affinity, respectively.Similar trends, often described as an inverted "U" have been reported for PFAS-BSA binding behavior 27 , with, for example, PFHxS exhibiting the highest K a and PFDA the lowest K a for the compounds plotted.The trend substantiates previous results that report that PFAS chain lengths greater than C 8 (i.e., greater than PFNA or PFOS) do not "fit" as well into the BSA binding pocket compared to shorter PFAS.Octanoic acid and decanoic acid both have smaller values of Log K ow than PFOA and PFDA and the same carbon chain length, respectively.When comparing PFOA and OA, the higher hydrophobicity likely drives the higher association constant for PFOA.On the other hand, PFDA is larger than DA arising from its fluorinated carbons and its size hinders its binding to BSA.
PFAS that bind strongly to BSA have been detected in cattle muscle and cow's milk, suggesting that protein binding is a driver of PFAS transport and excretion.Kowalczyk et al. 8 determined the percent of an ingested PFAS dose that is excreted in cow's milk and stored in muscle for PFOS, PFHxS, PFOA, and PFBS.Their study indicated that PFOS transfers more efficiently into cow's milk than PFOA or PFBS, which is consistent with PFOS having a higher K a (Table 2).

Binding cooperativity as evidenced by the Hill adsorption model
Neither the equilibrium-based model nor the Stern-Volmer model account for (1) the fluorescence of the protein-ligand complex and (2) binding cooperativity.These models assume a 1:1 binding event, which is not necessarily the case for PFAS-BSA but is a convenient simplification.At high concentrations of PFAS relative to BSA, more than one PFAS may bind to the protein.Thus, the Hill adsorption model was used to reflect binding cooperativity over the range of temperatures studied.For this analysis only PFAS are considered.The ability to The association constant obtained via the Hill model (Fig. 4) demonstrates the dependence of binding on temperature.An increase in K Hill with temperature reflects a binding process where the entropy gain associated with water desolvation of the PFAS is greater than the reduction of enthalpic interactions 18 .This is observed for all PFCAs except for GenX, where the entropic and enthalpic changes are balanced and K Hill does not change with temperature (Fig. 4A,B).PFOA, PFNA, and PFDA show the largest entropy gains based on K Hill increasing with temperature, consistent with their longer tails requiring a greater water solvation cavity (and greater  www.nature.com/scientificreports/entropy when that cavity is no longer required due to protein binding).For PFASs, protein binding based on K Hill did not exhibit temperature dependence.PFOS might be expected to show a similar trend to PFNA given its C 8 fluoroalkyl tail, but the low K Hill is consistent with the low K a (Table 2) and minimal shift T m (Fig. 2B).Differences in the temperature dependence of PFNA and PFOS could be due to many factors intrinsic to the PFAS (e.g., headgroup), to the protein (e.g., binding site location), and the subsequent intermolecular interactions.Considering binding cooperativity (Fig. 4C,D), when n Hill is less than 1 the binding of one ligand negatively impacts the binding of additional ligands.When n Hill is equal to 1, there is no effect and when n Hill is greater than 1, cooperativity is positive and the binding of one ligand positively influences the binding of additional ligands.For all PFAS n Hill is greater than one, indicating positive binding cooperativity.This result provides valuable insight, especially into the behavior of PFHxS, when combined with the other analyses.PFHxS showed a higher value of K a and K Hill than PFOS in the equilibrium-based model despite being less hydrophobic.The high degree of binding cooperativity may account for the mechanism driving these seemingly disparate results.When PFHxS binds it cooperatively increases binding of additional ligands, leading to a larger association constant.PFDA is another interesting case; its K a was low due to steric hinderances within the binding site but K Hill and n Hill were the largest of the PFCAs.This may reflect BSA quenching caused by non-specific PFDA adsorption outside of the binding pocket.Using fluorine nuclear magnetic resonance spectroscopy, we have previously reported that PFAS can adsorb non-specifically and that long PFAS (PFNA) may form hemimicelles on the protein surface.PFDA would be expected to exhibit even greater adsorption, perhaps enhanced by high binding cooperativity.
This work presents a new, rapid approach to analyze DSF data to aid in the rapid measurement of PFAS binding to BSA.By combining the equilibrium-based model at the melting temperature and the Hill adsorption model, valuable insight can be gained in terms of the binding mechanisms.While the equilibrium-based model offers a way to determine binding based on protein melting, the Hill adsorption model can provide binding and cooperativity constants over a range of temperatures.The understanding of temperature-based binding constants can aid in PFAS remediation and could inform pasteurization processes.In this case temperatures well beyond the melting temperature of PFAS:BSA complexes would be needed to desorb PFAS.The kinetics of BSA denaturing must also be consider.Times of half-conversion (folded to unfolded BSA) of 13.6 min and 1.5 min have been reported at 65 °C and 80 °C, respectively 38 .As specified by the USDA 39 , vat pasteurization at 63 °C for 30 min or high heat pasteurization at 88 °C for 1 s would partially desorb PFAS depending on the PFAS species and how it stabilized the protein and increased the melting temperature, but could be insufficient for significantly reducing the amount of bound PFAS.

Material
Bovine serum albumin (lyophilized powder, 99% fatty acid free) was obtained from Sigma-Aldrich (St. Louis, MO).A BSA concentration of 10 μM in pH 7.4 phosphate buffered saline (PBS) was used in each experiment.The solution of BSA was kept at 4 °C prior to use.

Differential scanning fluorimetry
Experiments were conducted using a Tycho NT.6 differential scanning fluorimeter (NanoTemper, Munich, Germany).PFAS was added to 2 mL of the stock BSA solution to obtain the PFAS:BSA or FA:BSA ratios of 0:1, 0.5:1, 1:1, 2:1, 3:1, and 4:1.Solutions were prepared a day in advance of the experiment and stored at 4 °C.Immediately prior to analysis, each sample was loaded into a 1 mm diameter glass capillary and placed in the instrument.Thermal scans were conducted from 35 to 95 °C at a scan rate of 0.5 °C s −1 with a 285 nm excitation wavelength.Fluorescence intensity was measured at 0.1 °C intervals at emission wavelengths of 330 and 350 nm.The ratio of the intensity at these wavelengths, I 350 /I 330 , was used to account for blue shifts in the emission peak wavelengths with increasing temperature.The minimum value of the first derivative of the I 350 /I 330 curve with respect to temperature, denoting the midpoint of the melting transition where the fraction of folded to unfolded protein is 1, was taken as the melting temperature T m .Each experiment was performed in triplicate.The same procedure was used for the control ligand warfarin.

Melting point association constant
As described by Eftink and Ghiron 44 , baseline subtraction of the raw fluorescence intensity data collected at 350 nm was performed at both high and low temperatures.The plots were then normalized by dividing by the maximum intensity value.These data were fit to Eqs. ( 1) and ( 2) to obtain a value for K d which is the inverse of the association constant (K d = K a −1 ) 29 .
(1) www.nature.com/scientificreports/ The term f U is the fraction of unfolded protein based on the concentration of unfolded protein, [U], divided by the total protein, [P] t , which is the sum of [U], folded protein [F], and folded protein with bound ligand [FL].
[L] is the total free ligand by correcting the total ligand concentration, [L] t , which is a main benefit of this model.K U represents the equilibrium between folded and unfolded protein, and K d is the ligand dissociation constant from the bound to unbound states.

Hill adsorption model
The Hill model has been used to correct for the presence of a fluorescent protein-ligand complex and binding cooperativity 21,34 .I 0 and I are the fluorescence intensity of BSA in the absence and presence of the ligand and I res accounts for residual fluorescence at infinite quencher concentration.K Hill and n Hill are the Hill binding constant and the cooperativity constant, respectively, and [Q] is the concentration of added quencher (PFAS or FA). (2)

Figure 1 .
Figure 1.Fluorescence intensity (I) from 35 to 80 °C depicting ligand quenching of BSA.The BSA concentration in pH 7.4 PBS buffer was constant at 10 μM.PFAS:BSA ratios ranged from 0:1 to 4:1.Panes (a-c) are specific to PFOA and demonstrate the vertical transit at 37 °C from which a (b) Hill model plot can be created to calculate K Hill and n Hill , and (c) the folded to unfolding transition from which the protein melting temperature, T m , can be determined.Panes (d-f) are the quenching curves for NA, PFNA, and PFOS, respectively, and compare the effects of headgroup and alkyl or fluoroalkyl tails for a common number of tail carbons.

Figure 2 .
Figure 2. The change in melting temperature upon addition of PFAS or fatty acid to BSA as a function of the ligand:protein ratio.Filled symbols indicate PFAS compounds and open symbols indicate fatty acids.Carboxylic acids are shown in (A) and sulfonic acids in (B).Each experiment was performed in triplicate and error bars not visible are smaller than the symbols.

Figure 3 .
Figure 3. PFAS-BSA association constants (K a ) for PFAS and fatty acids determined near the observed melting temperature of BSA (61 °C) based on the equilibrium-based binding model plotted against (a) Log Kow and (b) the number of fluorinated or hydrogenated carbon atoms in the PFAS or FA.